Prokaryotic Expression and Enzyme Activity Analysis of Histone Deacetylase MrSir2 from Monascus ruber
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Abstract:
According to the sequenced genomic DNA information, the whole cDNA sequence of mrsir2 gene was obtained by rapid-amplification of cDNA ends (RACE) corresponding to coding sequence (CDS) with 1539 bp, which codes for 512 amino acids and contains a conserved domain of SIR2 protein. The CDS of mrsir2 was optimized according to the codon preference of E. coli. The CDS of mrsir2 was ligated to the pET-28b vector and then transferred into the host strain E. coli BL21 to be induced expression by IPTG. The expression condition was optimized. The experiment results showed that the target protein MrSir2 was expressed well in soluble way when the host was induced by IPTG at a final concentration of 0.25 mM for 16 h at 16 °C. The soluble recombinant protein was purified with Ni2+ column affinity chromatography. Result of SDS-PAGE showed a band with the molecular weight of approximate 75 ku.The protein concentration was as high as 1.97 mg/mL. The target protein was identified by western blot. The enzyme activity was 78.5 (OD/min/mg) detected by Epigenase? Universal SIRT Activity/Inhibition Assay Kit (Colormetric). The inhibition rate of 780μM dihydrocoumarin(DHC) to MrSir2 was 47%. The soluble expression of MrSir2 provided materials for understanding its enzymatic characteristics. Results will provide the foundation for exploring the interaction of protein-protein in vitro.
